This project is focused on understanding clathrin-independent forms of endocytosis (CIE). Endocytosis that occurs without clathrin coats occurs in all cells but is poorly understood. We are interested in studying the cargo proteins that enter cells by this mechanism, their intracellular itinerary once they have been internalized and whether they contain amino acid sequences that allow for specialized sorting within cells. We have been identifying new cargo proteins and found that a subset of these proteins take alternative traffic routes once they have entered cells. The major histocompatibility complex Class I protein (MHCI), is a prototypical clathrin-indepenent cargo protein and after internalization it reaches endosomes that contain cargo proteins such as the transferrin receptor that enter via clathrin-depenent endocytosis. From there, MHCI travels either to late endosomes and lysosomes where it is degraded or on to recycling tubules that return MHCI back to the cell surface. CD44, CD98, and CD147, however, show an altered itinerary in many cells where they traffic directly into the recycling tubules and avoid trafficking to lysosomes. Consistent with this altered itinerary, CD44, CD98 and CD147 are long-lived proteins and are not degraded like MHCI, which is routed to lysosomes. One of the functions of CIE may be to accumulate select group of cargo proteins in endosomes for specific functions. We recently showed that during vasculogenesis, using a model of lumen formation in human vascular endothelial cells, that CIE cargo proteins are delivered to these forming lumens, suggesting that CIE endosomal membranes may contribute to cell hollowing during vessel morphogenesis (Porat-Shliom and Donaldson, 2014). The altered trafficking followed by CD44, CD98 and CD147 allows these proteins to avoid transfer to lysosomal compartments and thus avoid degradation. Other CIE cargo proteins, such as MHCI, traffic to lysosomes and we have been interested in the sorting of cargo at the sorting endosome and understand how MHCI is tagged for degradation. With that goal in mind we set up a system for following the turnover of model cell surface proteins using the interleukin 2 receptor alpha subunit (Tac) coupled to the SNAP tag as our model system (Cole and Donaldson 2012). The turnover of various SNAP-Tac variants demonstrated that the half-life of the proteins was determined by their mode of entry (clathrin-mediated vs clathrin-independent), orientation in the cell membrane (i.e. integral membrane vs. glycosylphosphatiylinositol-anchored proteins) and the capacity for ubiquitination of residues on the cytoplasmic tail (Karabasheva et al 2014). Additionally, a striking and efficient shedding of SNAP-Tac into the media was observed when O-linked glycosylation was inhibited, demonstrating that ectodomain shedding may play a significant role in turnover of cell surface proteins. We are interested in the role that ubiquitin modification of cargo proteins plays in regulating the sorting of cargo and routing of cargo to lysosomes for degradation. We previously showed that over expression of the MARCH8 E3 ubiquitin ligase leads to ubiquitination and degradation of CD98, normally a long-lived protein (Eyster et al 2011). We now show that expression of a de-ubiquitinase (DUB) or ubiquitin-specific protease (USP) can counteract the effect of expression of MARCH8. This DUB is called TRE17/USP6 and was previously shown to be unregulated in ABC bone cancer. Expression of TRE17 specifically de-ubiquitinates CD98 and returns its trafficking to the normal pathway (i.e. avoidance of lysosomes) (Funakoshi et al, 2014). However TRE17 did not rescue the effect of MARCH8 on degradation of the transferrin receptor.